This proposal describes the use of novel proteomic methods and classical pharmacology to characterize the topology of the dopamine transporter (DAT) and identify and characterize post translational modifications (PTMs) conserved between the human and rat DAT. This proposal outlines research to complete three specific aims: 1) Characterize the topology and PTMs of the human DAT (hDAT) expressed in HeLa cells. 2) Characterize the topology and PTMs of the rat DAT (rDAT) expressed in HeLa cells and the PTMs of rDAT in rat straitum. 3) Correlate the function of the rDAT and hDAT with conserved PTMs. These three aims will be accomplished using proteomic methods that allow for the digestion of the exposed, protease-accessible domains of the dopamine transporter while it is embedded in its native, cellular environment. This digestion will create two sets of peptides: exposed protease-accessible peptides which correspond to the cytoplasmic and extracellular domains of DAT; and membrane-embedded peptides which correspond to the integral membrane domains of DAT. Both sets of peptides will be analyzed using mass spectrometry to identify the peptides. These two analyses will allow for a comprehensive topological characterization of the transporter and give a more complete structural picture of the transporter and how the residues involved in binding and transport are placed with relation to the plasma membrane. Simultaneously, mass spectrometry and 2D gels will be used to identify PTMs on DAT. Newly identified PTMs conserved between hDAT and rDAT will be quantified by mass spectrometry using synthetic isotopically labeled peptides, and characterized by creating DAT mutants without that PTM site and performing uptake and binding assays on the mutants and assaying for surface expression. Relevance to Public Health: The dopamine transporter is an important target for common drugs of abuse, such as cocaine and methamphetamines; it is also an important therapeutic target for illnesses such as Parkinsons Disease (PD) and Attention Deficit Hyperactivity Disorder (ADHD). By characterizing its structure and modifications, important insights will be gained into its function and regulation, which can be used to build highly specific and sensitive Pharmaceuticals for aiding in 1) recovering from drug addiction and 2) treatment of disease. [unreadable] [unreadable] [unreadable]